Arrhythmia termination using Global Optogenetic Stimulation in ChR2 mice hearts

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Arrhythmia termination using Global Optogenetic Stimulation in ChR2 mice

hearts

Dissertation

for the award of the degree

“Doctor rerum naturalium”

of the Georg-August-Universit¨at G¨ottingen

within the doctoral program Physics of Biological and Complex Systems of the Georg-August University School of Science (GAUSS)

submitted by

Ra´ul Alejandro Qui˜nonez Uribe from Ensenada, Mexico

G¨ottingen, 2020

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Thesis Committee

Prof. Dr. Stefan Luther, Research Group Biomedical Physics, Max Planck Institute for Dynamics and Self-Organization

Prof. Dr. Andr´e Fiala, Dept. of Molecular Neurobiology of Behaviour, University of G¨ottingen

Prof. Dr. J¨org Enderlein, III. Institute of Physics, University of G¨ottingen

Members of the Examination Board Referee: Prof. Dr. Stefan Luther 2

^nd

Referee: Prof. Dr. Andr´e Fiala

Further members of the Examination Board:

Prof. Dr. J¨org Enderlein

Dr. Andreas Neef, Campus Institute for Dynamics of Biological Networks, Max-Planck-Institute for Experimental Medicine

Prof. Dr. Luis A. Pardo, Dept. of Molecular Biology of Neuronal Signals Max Planck Institute for Experimental Medicine

Brett Carter, Ph.D., Synaptic Physiology and Plasticity, European

Neuroscience Institute

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Dedication

Para mi mam´a, mis hermanos y la familia. Porque el tiempo vivido fuera de Ensenada ha sido tiempo lejos de ustedes.

To my mom, my brothers and family. Since the time spent away from Ensenada has been time spent away from you.

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Acknowledgments

First of all, I would like to thank my supervisor Stefan Luther for giving me the opportunity to join the Biomedical Physics research group more than 4 years ago and for guiding me throughout my doctoral project and for enthusiastically and generously sharing his knowledge in pursue of my development as a researcher. I would also like to thank Claudia Richter for teaching me plenty of skills and subjects from the laboratory and from the field of cardiac optogenetics. Also, thanks a lot for the great help in writing the paper.

Special thanks to my Thesis Advisory Committee Jörg Enderlein and André Fiala for interestingly attending the meetings and thoughtfully evaluating my progress, providing me with their knowledge and giving me feedback when necessary. Also to my Examination Board Jörg Enderlein, André Fiala, Andreas Neef, Luis Pardo and Brett Carter for showing interest in my research accepting the invitation to my thesis defense, as well as for the interesting questions they will ask on this day.

I would like to thank my colleagues for making the experience of working at this research group very smooth, interesting and friendly. Thanks a lot for helping, for the discussions and for the different activities organized. From everyone I learned about science and about life and culture. Old and new colleagues I would like to thank: Jan Christoph, Tariq, Henrik, Sebastian B., Sebastian S., Svetlana, Laura, Thomas, Johannes, Filippo, Florian, Baltasar, Justine, Jan L. Special thanks to Vineesh for sharing the Be-Optical experience together and to Sayedeh for sharing many talks on cardiac optogenetics and doctorate life.

I would also like to hugely thank Marion and Tina. Without all your help this thesis would not have been possible. You helped me many times with different situations and definitely made working at the laboratory a lot easier. I would like to thank Annette, Claudia and Janna for the help provided in the editing of this thesis. Special thanks to Annette for the many comments that helped improve the quality of my writing.

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Abstract

Cardiac arrhythmias represent a health threat worldwide that can end in sudden death. Treat- ments which include electrical shocks, anti-arrhythmic drugs and ablation show various disadvantages such as discomfort for the patients, side and pro-arrhythmic effects as well as lack of specificity. Moreover, arrhythmias display complex spatio-temporal behaviors making their study a challenging task. Cardiac optogenetics is an emerging field used to enable photo-control of cardiomyocytes by inscribing in them light-sensitive ion channels such as Channelrhodopsin-2 (ChR-2). Optogenetic stimulation offers a new dimension to investigate cardiac electrophysiology, from high spatial and temporal resolution to cell specificity and intensity dependent effects. Previous studies have shown the capability of arrhythmia control and termination using optogenetics in ChR-2 transgenic mice hearts. However, these studies relied on local stimulation, which requires either high intensities or long pulses. In this doctoral thesis I seek to investigate the advantages of globally illuminating the heart as a method of optogenetic cardioversion. In order to do so, I first characterized the attenuation of light by the cardiac tissue as well as the response to light stimulation of our model of ChR-2 transgenic mice hearts using the Langendorff-perfusion technique. The effect of the intensity, pulse width and diameter of the fiber under different experimental conditions involving tyrode, blebbistatin and the voltage sensitive dye Di-4-ANBDQPQ were investigated in this first step.

Next, I designed an experimental setup that allowed global illumination of the isolated heart.

Arrhythmia induction was facilitated using the drug pinacidil before stimulating the hearts using pulses of varying intensities and lengths. I also analyzed the efficiency of optogenetic cardioversion to different arrhythmia wave morphologies, and lastly investigated the time it takes an arrhythmia to be terminated using light in order to better understand the mechanisms behind this phenomenon. My results show that both intensity as well as the length of the pulse affect every aspect of optogenetic stimulation. With a higher effect from intensity, increasing these parameters leads to a higher success in pacing and cardioversion and shortens the time required to terminate an arrhythmia. Furthermore, optical mapping analysis allowed the visu- alization of spatio-temporal electrical waves on the heart surface. It could be shown that most arrhythmias were terminated by light stimulation of the excitable gap which caused the collision of the arrhythmic wave. The results obtained improve the understanding of optogenetic cardioversion from different perspectives and offer a head start in the design of experiments using large animal models with aims on a future clinical translation.

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Abbreviations

AP Action Potential

APD Action Potential Duration ChR2 Channelrhodopsin-2 LED Light Emitting Diode LV Left Ventricle

MAP Monophasic Action Potential ms milliseconds

MVT Monomorphic Ventricular Tachycardia mW/mm² milliwatts per millimeter square

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1. Introduction

Covering cardiac electrophysiology, image analysis, physics and other fields, “Arrhyth- mia termination using Global Optogenetic Stimulation in ChR2 mice hearts” is a multidisci- plinary work performed during my years of PhD training. The keywords in the title of this dissertation are heart, arrhythmias and optogenetics. Using these three words my PhD project can be explained as follows.

As we know the heart is the blood pump of the body and it functions via electrical impulses. An abnormal beating or function in the electrical propagation of the heart is called an arrhythmia. Electrical stimulation as given by pacemakers or defibrillators can force the heart to a normal rhythm. I used a new technique called optogenetics that allows stimulation of the heart using light as a harmless alternative to electrical stimulation. Naturally, hearts are not sensitive to light, thus transgenic mice hearts were used to perform experiments. These hearts expressed the light-sensitive ion channel Channelrhodopsin-2 (ChR2) that allows the photo- control of the membrane potential of cardiomyocytes. Therefore the word “optogenetics”.

Optogenetic cardioversion has been successfully demonstrated by different groups [12, 61, 19]. However, the mechanisms underlying optogenetic cardioversion remain largely elu- sive. In order to further understand the requirements and factors behind successful optogenetic arrhythmia termination I hypothesize that utilizing global epicardial illumination would lead to a decrease in both light intensity and pulse width necessaries to stop an arrhythmic activity on the heart and at the same time help us understand the events involved in this task.

The dissertation is divided into three parts, which are at the same time composed by chapters. The first part reviews the topics necessary to understand the work done. Here the reader can learn about the basics in order to understand the project, including the normal functioning of the heart, arrhythmias, the principles of optogenetics as well as its application on the cardiac field. The first part ends with a chapter describing the methods.

Part two constitutes the results; starting from the characterization of the response of the heart to optogenetic stimulation before moving into the investigation of a more complex task such as optogenetic cardioversion and lastly performing a deeper look into how optogenetic cardioversion works. Since each chapter has different research objectives, an introduction, results, discussion and conclusions section was included to each one. This way the reader can identify the corresponding meaningful information in a focused and accessible manner.

Chapter 7 wraps the most important outcomes of my doctoral work in a published article

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1. Introduction

inFrontiers in Physiology. “Energy-Reduced Arrhythmia Termination Using Global Photo- stimulation in Optogenetic Murine Hearts” describes the first results obtained with the global stimulation setup I assembled with the advice of my supervisors. These first experiments paved the way for more interesting questions and experiments that are investigated in chapters 8 & 9, such as the effects of arrhythmia morphology on cardioversion success and the time required to terminate arrhythimias using global illumination.

Lastly, part three closes the thesis with a general discussion of the project describing different outlooks and challenges emerging from this research work and the conclusions obtained.

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Part I.

Scientific Background

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2. The Heart

2.1. The electromechanical pump

The human heart is a muscular organ composed of four chambers, working synchronously to pump oxygenated blood coming from the lungs into the circulation and non-oxygenated blood into the lungs coming from the system [24, 32].

Figure 2.1.:Blood flow through the heart.Blood low on oxygen is transported from the sys- temic circulation and into the right atrium before being pumped from the right ventricle (RV) into the lungs. Freshly oxygenated blood returns to the heart through the pulmonary veins and into the left atrium. (Continues on next page.)

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2. The Heart

Figure 2.1.: Then the atrium contracts pushing the blood into the left ventricle, which is the last location before its delivery back into the circulation ejected by the pressure formed due to the contraction of the ventricular walls. In summary, the right side of the heart is in charge of forcing the blood to the lungs for its oxygenation, and after that, the left side forces the blood to the rest of the body for its distribution.

Obtained and modified from [99, 32].

The superior and inferior vena cava transport blood that is low in oxygen, coming from the upper body and from the trunk and legs, respectively. This blood flows into the right atrium, and then is pumped by the atrium through the tricuspid and into the right ventricle, which will be at the same time in charge of pumping it to the lungs. In the lungs, the blood will go through the process of oxygenation before returning to the heart through the pulmonary veins. It will reach the left atrium and then the left ventricle (LV). Due to the ventricle’s contraction, the pressure will rise until it opens the aortic valve and finally gets ejected into the circulation [24, 32] (Fig.2.1).

The coordinated contraction and relaxation of the four chambers of the heart is ruled by its electrical activity. Therefore, arrhythmic behavior can lead to a variety of cardiovascular diseases. In order to understand the underlying mechanisms leading to the heart’s function and malfunction it is important to know how electricity is generated and distributed in and around the heart from molecular to organ level [24, 32].

2.2. Electrophysiology of the heart

The heart is composed of millions of cardiomyocytes [48], specialized cardiac cells.

Each cardiomyocyte is a living system on its own, composed of different proteins that synchronized generate changes on the voltage potential of the cell membrane. These changes lead to the contraction of the cell and at the same time to the ordered contraction of the chambers of the heart and ultimately to the pumping of the blood from the heart and into the body’s supply system.

2.2.1. The Cardiac Conduction System

The generation and propagation of electrical signals in the heart follows a specific pathway in order to achieve a synchronous and ordered electrical excitation and further contraction of the heart. The atrial and ventricular myocytes, compose the major working force and also posses the ability to conduct electrical impulses.

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2.2. Electrophysiology of the heart The cardiac conduction system is a network comprised of both pacemaker cells, which are in charge of periodically generating electrical signals [51], and conduction cells, which are responsible of transporting the signals (bundle of His, left and right bundle branches, and Purkinje fibers). This way the electrical excitation is generated and spread in a fast and highly ordered manner.

Figure 2.2 illustrates the cardiac conduction system. The signals originate in the sinoatrial (SA) node since it hosts the self-excitatory pacemaker cells. The SA node is located in the upper part of the right atrium. Therefore, the SA node and its cells are responsible for the heart beating frequency. A depolarizing current unique to these cells allows them to rhythmically generate action potentials. The electrical excitation generated by the action potentials is spread to the neighboring cells via specialized connections between cells, the gap junctions. The electrical wave travels first through right atrium and then through the left, con- tracting almost simultaneously. From there, the impulse is transported via junctional fibers to the atrioventricular (AV) node, which is located at the bottom of the right atrium, above the interventricular septum [34, 65, 81, 80].

Figure 2.2.:The cardiac conduction system. The normal conduction starts at the sinoatrial node and excites the atria before reaching the atrioventricular node. After reaching the bundle of His, the pathway is branched into the right and left ventricles. The differences in the intrinsic pacemaker rates allow normal excitation to occur and if the SA becomes inactive, then the next structure on the activation pattern would generate the spontaneous rhythm. Image obtained with permission [81].

The main task of the atrioventricular node is to relay the electrical signal coming from the SA node to the ventricles. A delay in the signal transmission is also introduced through the AV junction, while conducting the impulses to the bundle of His. This delay has a functional

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2. The Heart

purpose in the pumping action of the heart, allowing the atria to contract completely and empty the blood into the ventricles before these start to contract.

From the bundle of His the depolarization wave is bifurcated into the right and left bundle branches. The last transmission elements before reaching the ventricles are the Purkinje fibers, which conduct the impulse at a high speed and spread it to different points of the left and right ventricular walls. Lastly, the ventricular myocardial depolarization will occur from endocardium to epicardium and from apex to base, serving the pumping mechanics of the ventricles [34, 65, 81].

All of these electrical excitation events lead to a contraction, therefore the specific order and synchronization are crucial in order to pump the blood in an efficient and sustainable manner. However, synchronicity does not start at the structure level. The generation and propagation of electrical impulses and at the same time the contraction of the tissue start from activities at cell level. The next section is dedicated to understanding how an an electrical impulse generates.

2.2.2. The Cardiac Action Potential

Membrane potential and ion channels

Cardiomyocytes are electrically excitable cells. The selective permeability of their membrane keeps the inner and outer concentration of ions constantly at different levels, creating a transmembrane potential. The excitation of the membrane by electrical impulses changes its voltage. These changes lead to the opening of voltage-sensitive ion channels that allow a quick flow of different ions inside and outside the cell, altering the permeability of the cell and depolarizing it. Ultimately, the change in membrane potential is followed by the cell contraction. Membrane excitability of the cell will depend on different ion channels, pumps and transporters located through its membrane [32, 81]. In this section we will go through the most important actors and events that lead to the excitation and contraction of cardiomyocytes.

Ion channels are a group of protein molecules located in the cellular membrane. More specifically, cross the membrane from the outer part into the inner part in order to create a route of transport for ions and other specific molecules. Ion channels are distinguished by two important features: 1) They normally have a selective transportation, allowing only certain ions to travel across the membrane, and when they are open the ions move by diffusion along the channel due to the difference in concentrations of the specific ion inside and outside the cell. This kind of transportation along the membrane is defined as passive. 2) Many of these channels can open and close. These processes are regulated by either electrical signals (channels activated by a voltage threshold), or chemical signals (activated by ligands). The three types of ion channels that play the most important roles in cardiomyocyte electrophysiology

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2.2. Electrophysiology of the heart

are sodium, potassium and calcium, and they are all regulated by electrical signals [32, 81].

Myocardial cells hold a resting membrane potential of approximately −90 mV. This potential results from the separation of anions and cations across the membrane and is mainly determined by the following factors: a) inner and outer concentration of charges (ions), b) permeability of those ions through the channels, and c) the activity of ion pumps.

Typically, in mammalian myocytes, the concentration of sodium ions (Na⁺) is higher outside the cell at approximately 140 mM compared to an intracellular concentration of 5 mM to 34 mM. In a similar way, calcium (Ca²⁺) will have an extracellular concentration of approximately 3 mM, compared to an almost negligible amount inside the cell. Contrary to these cations, the concentration of potassium (K⁺) will be 104 mM to 180 mM intracellularly with just 5.4 mM outside the membrane. These three ions play a critical role in maintaining the membrane potential at rest and also in inducing the changes in voltage during the action potential [34].

Action Potential

An action potential (AP) is triggered when the membrane potential is shifted towards a more positive value of approximately−60 mV due to a depolarizing current normally induced by neighboring cells through gap junctions. If this stimulus is not large enough to reach the threshold potential, no depolarization of the cell will occur and therefore, no action potential will be triggered [81].

If the threshold is reached, a series of opening and closing of different channels will take place, depolarizing and afterwards repolarizing the cell back to its resting potential. There are five phases identified during an action potential (Fig.2.3) [33, 81, 34, 59]:

Phase 0 - Rapid Depolarization: The stimulus that depolarizes the cell to the threshold ac- tivates the voltage-dependent sodium channels (Nav), abruptly changing the permeability of the membrane to Na⁺. Due to the difference in concentration, there is a rapid influx of Na⁺ into the myocyte depolarizing the cell to positive voltages of about 20 mV, almost exclusively generated by the movement of Na⁺ cations. The voltage-gated Na⁺ channels will inactivate within milliseconds from opening and the permeability of the membrane to Na⁺ will then again be decreased. At the end of this phase and with a delay compared to the Na⁺ channels, the voltage-sensitive Ca²⁺channels will also start to open, increasing the permeability of Ca²⁺. At the same time the increased amount of intracellular calcium also induce the opening of Ca²⁺channels inside the cell, at the sarcoplasmic reticulum. This process is called “calcium induced calcium release” and marks the beginning of the contraction process.

Phase 1- Early Repolarization: Together with the closing of the Na⁺channels, there is a small repolarization caused by the opening of K⁺ channels, creating an outflow of K⁺ ions and

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2. The Heart

Figure 2.3.:Action potential of cardiomyocytes. The action potential is divided into five different phases, leading to the various changes in the transmembrane potential (TMP). Phase 0 is characterized by the rapid depolarization caused by the opening of voltage-dependent sodium channels. In Phase 1, a small repolarization takes place due to the efflux of potassium before the plateau phase (Phase 2) where a balance of inward Ca²⁺current and K⁺outward currents keeps the voltage stable.

At last, Ca²⁺channels close in Phase 3, leading to a repolarization back to negative values by the continuous K⁺currents. During Phase 4 the membrane potential is stable at the resting state until another stimulus depolarizes the membrane up to the threshold. Image obtained from [35].

reducing the membrane potential to approximately 0 mV. Inactivation of the Na⁺ channels can happen as soon as 1 ms after their opening and excitation of the cell will not be possible until the majority of the channels recover. Therefore, these channels are the main determinants of the excitability of the cells of each specific region of the heart.

Phase 2 - The plateau phase: Due to the outflow of potassium and the inflow of calcium through L-type calcium channels, a plateau phase is reached, where the voltage is stable around 0 mV for a period of a couple of hundreds of milliseconds. L-type calcium chan-

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2.2. Electrophysiology of the heart nels inactivate at a slower pace compared to other voltage activated channels such as the Na_v and are therefore important contributors to the plateau phase. As mentioned in Phase 1, the influx of Ca²⁺ triggers the release of Ca²⁺ from intracellular compartments, leading to the contraction of the cell.

Phase 3 - Repolarization: This phase ends the action potential. The gradual inactivation of Ca²⁺ channels, together with the K⁺outward currents bring the membrane potential back to its negative values. Moreover, the activity of different ion pumps returns the sodium and calcium ions outside of the cell and potassium ions to the interior.

Phase 4 - The Resting Phase: This is the original phase of the cell before receiving a new stimulus and triggering an action potential again. In this phase Na⁺and Ca²⁺ channels are closed and a different K⁺current helps maintaining the negative membrane potential. However, Na_v channels will need time to recover before gaining the ability to be excited again. This time is called refractory period.

Therefore, during the refractory period cells cannot be excited. It is the interval of time from depolarization to recovery of excitability, and it is related to the action potential duration.

Differences in the refractory period in adjacent regions can aid the generation of arrhythmia [14]. Arrhythmia and arrhythmogenesis will be explained in Chapter 3.

The expression of ion channels as well as their properties will be different for each structure of the heart (sinoatrial node, atrioventricular node, atria, ventricles) and will also vary for different animal species. Therefore, cardiac action potentials have different waveforms depending on their location. Those differences will contribute to the normal propagation of excitation waves through the heart. Fig.2.4 A illustrates the waveforms for the excitation pathway described before.

Fig.2.4 B & C show the differences between human and mouse action potentials. The latter is shorter and is lacking a plateau phase. These two characteristics can be determined by a smaller expression of Ca²⁺ channels on the cell membrane compared to the human heart and by a larger expression of K⁺. The differences lead to a faster repolarization of the membrane potential and shorter action potentials (30-80 milliseconds [ms] compared to 150 ms to 400 ms).

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2. The Heart

Figure 2.4.:Differences in action potentials. (A)Regional differences in the action potential waveform, and how the regional electrical signals form up the electrocardiogram (ECG).(B)Human and(C)mice action potential waveform. Images obtained and modified with permission from [59, 33].

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3. Cardiac Arrhythmias

Cardiac arrhythmias are defined as an abnormal rhythm of the heart, which in other words would be abnormal electrical activity in the heart. As discussed in Chapter 2, the heart follows an ordered electrical excitation automatically starting at the sinoatrial node and ending at the ventricular myocardium. A disruption of the frequency, origin or route of the electrical propagation can cause or be considered an arrhythmia.

There exists a variety of types of arrhythmias, with also different ways to classify them.

For example by heart rhythm, where a bradycardia is termed for a slow heart rate, tachycardia for an abnormally fast heart rate, and during fibrillation the pace, amplitude and morphology are not constant [81, 72]. Arrhythmias can also be classified depending on the region of the heart where they take place. Supraventricular tachycardia originate in the atria and ventricular arrhythmia in the ventricles. An additional classification is by the morphology they show in an electrical recording such as the electrocardiogram, separating them into monomorphic and polymorphic arrhythmias [82].

When the heart is suffering an arrhythmia, it is unable to properly pump blood from its chambers and into the circulation. Sudden cardiac death is one the leading causes of natu- ral deaths in the United States and Europe and approximately half of them are attributed to ventricular arrhythmia [37].

3.1. Arrhythmogenesis

A healthy heart is able to maintain its function without any anomalies. Arrhythmias indicate abnormal electrical activity of the heart. The causes of an arrhythmia are broad and they commonly represent a multiscale problem that can originate from one or many of the different scales the heart functions: molecular, cellular, tissue, structures, chambers. Alter- ations in function of the ion channels can promote arrhythmia initiation, and so can changes in the conductivity of a specific region of the ventricle caused by ischaemia. Nonetheless, the abnormal electrical activity, leading to the initiation of arrhythmias can present in two forms:

a) abnormal impulse generation which are triggering events, or b) abnormal impulse conduction, such as heterogeneities or changes in the substrate that affect the pathway of conduction and can lead to reentry. Both of these mechanisms on their own or combined can provoke the

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3. Cardiac Arrhythmias

initiation of arrhythmias [66, 39, 23, 33]. In this section we will look into the mechanisms and focus on ventricular arrhythmias.

3.1.1. Triggering events

One of the most common mechanisms that trigger arrhythmias at cellular level are after- depolarizations (Fig. 3.1), which are abnormal impulses or extrasystolic membrane depolarizations (outside of the normal depolarization) that can lead to action potentials. These action potentials at the same time can ignite ectopic beats which may result in sustained tachycardia.

Figure 3.1.:Early after-depolarizations and delayed after-depolarizations. EADs take place during the repolarization (i) of an action potential, while DEAs arise once the cell is again repolarized (ii). They can both prompt extrasystoles. Image obtained with permission from [39].

Early after-depolarizations (EADs) occur during the repolarization time of a prolonged action potential (Phases 2-3). This allows the L-type Ca²⁺ channels to recover. As mentioned before, these channels are normally open during Phase 2 and are responsible of the plateau in the action potential. The delayed repolarization is commonly caused by a decreased outward potassium current, and if it enables enough time Ca²⁺ channels recover their excitability, re- open and produce a depolarizing current. This at the same time initiates a positive feedback on channel opening resulting in the after-depolarization and potentially triggering an action potential [66, 39, 23, 33].

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3.1. Arrhythmogenesis Delayed after-depolarizations (DEAs), different from EADs, occur after a full repolarization (Phase 4). They can be elicited from an enhanced Ca²⁺ release from the sarcoplasmic reticulum [66, 39, 23, 33].

Other examples of triggering events are alternans and automaticity. During alternans, cells present changes in the action potential duration, alternating long and short action potentials. Automaticity is when cells that normally do not depolarize on their own do so. Pace- maker cells of the sinoatrial node are able to periodically undergo spontaneous depolarization, and this is normal automaticity. Enhanced automaticity (increased rate), or automaticity acquired by other cells that are not functioning as pacemaker cells can lead to arrhythmia generation [82].

3.1.2. Heterogeneities or changes in the substrate (Reentry)

Tissue heterogeneities can lead to arrhythmias in different manners, and they are the basis of most arrhythmias in the clinic. Reentry refers to a circulating electrical wave in which an impulse travels repetitively around a region of the heart. The prerequisites for the development of reentry are: 1) an abnormal electrical circuit around an anatomical block, 2) an unidirectional block and 3) conduction velocity slow enough to allow recovery of excitability in time for reexcitation by the circulating impulse. The wavelength is defined as the product of the effective refractory period by the conduction velocity of excitable media such as cardiac tissue. If a on of the paths in a circuit bifurcation presents a decrease in wavelength an excitation loop in this circuit can be created. The path with lower conduction velocity forms a loop if the propagation is first blocked due to a longer effective refractory period, then a next wave could travel retrogradely along the path that was originally unexcitable creating the reentry circuit (Fig.3.2 A). This is called anatomical reentry and many times can give rise to a monomorphic ventricular tachycardia (MVT) [23, 66, 14].

An arrhythmia can also be triggered without an anatomical obstacle. Slowed conduction velocity due to a decreased membrane excitability or decreased cell-to-cell coupling may also result in slowed or blocked conduction. In a different scenario, a heterogeneous area on the tissue with a longer refractory period can also initiate an arrhythmia. An electrical wave originating in the periphery would not propagate through this specific area, but instead travel around it. Afterwards entering it from the opposite direction creating what is called a figure of eight reentry (Fig.3.2 B). In yet a different scenario, a region of cells with triggering activity surrounded by unequal dispersion of refractoriness could start a propagating wave only towards one direction but not to the other initiating a reentrant activity (Fig.3.2 C) [39, 14].

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Figure 3.2.:Arrhythmogenesis due to heterogeneities in the cardiac tissue. Caption on next page.

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3.2. Study of cardiac arrhythmias in isolated hearts

Figure 3.2.:Arrhythmogenesis due to heterogeneities in the cardiac tissue. (A) An anatomical reentry can be initiated when an anatomical block separates two pathways with different conduction velocities. After a wave originated by the sinus rhythm propagates across the two pathways (1,2) a second wave coming from an abnormal activity or source will not be able to travel through pathway 1 (light gray), since it has an increased refractoriness (3). Once the wave reaches the end of pathway 2 (white) it will be able to travel retrogradely (4,5) through pathway 1 initiating the re-entrant arrhythmia (5,6). (B)A region with longer refractory period can also initiate arrhythmia reentry when an unidirectional conduction block occurs in this region. An ectopic wave (white arrows) traveling after a sinus wave (1) would travel around the region presenting the heterogeneity (2,3) and then retrogradely into it (4) starting the arrhythmic pattern. (C) Reentry can also be induced when the same region of the cardiac tissue presents abnormal electrical activity (such as early afterdepolarizations, in this case) and heterogeneities in the propagation. After a normal excitation, an ectopic activity due to EAD would first excite the area with highest conductivity and then propagate. Images obtained and modified with permission from [44, 66]

Even though the mechanisms described before are the most common, there are other mechanisms based on the same principles that can start arrhythmias. In summary, a combination or one of the following is needed: a) an ectopic electrical activity or trigger, b) heterogeneities in the substrate such as a conduction block or differences in the effective refractory period or conduction velocity.

3.2. Study of cardiac arrhythmias in isolated hearts

3.2.1. Langendorff perfusion

The Langendorff perfusion is a technique to reanimate isolated hearts in vitro. It has been used to investigate a number of different physiological phenomena in the heart such as electrical activity, blood flow regulation, metabolism, effects of drugs, and others. One of the advantages of the technique are the study of the isolated heart without being influenced by other organs and systems from the body. This of course at the same time makes the results obtained harder to directly translate into a clinical scenario [5].

During the experiment the hearts are retrogadely perfused by cannulating the aorta, with the pressure of the perfusate closing the aorta and allowing it to flow through the coronary system [81]. This is an advantage especially for the study of cardiac arrhythmias since the electrical malfunction investigated will not lead to a decrease in perfusion of the heart since it

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will not depend on its own pumping to be perfused.

The heart is suspended in a prewarmed bath to keep it at optimal temperature. The perfusion flow can be controlled either via constant pressure or constant flow and during the experiment this and other physiological parameters such as the temperature, pH and electrical activity can be measured to keep track of the heart’s status. In order to keep the heart close to a physiological condition, the perfusate is oxygenated with 95% oxygen and 5% CO₂ and heated up to the necessary estimated temperature [5].

Once the perfusion setup is running it can be adapted to the specific needs of the study.

Since in this PhD work I am looking to understand cardiac arrhythmias, the optical mapping technique was also implemented for some experiments.

3.2.2. Optical Mapping

Optical mapping is a technique based on optical sensors which can be fluorescent dyes or genetically encoded and are used to track the transmembrane potential or intracellular calcium concentration of isolated hearts or 2D cardiac cultures. It is a contactless method that has improved the study and understanding of different electrophysiological phenomena such as cardiac arrhythmias. This is due to its ability to record action potentials from different regions of the heart simultaneously that result in the spatiotemporal mapping of travelling waves across the sample [63, 81].

In order to perform optical mapping of transmembrane voltage (implemented for this doctoral thesis), the fluorescent voltage sensor is loaded into the sample. Synthetic voltage sensors can be directly injected to the hearts using the Langendorff perfusion technique. Once the dye is loaded, a source light with the excitation wavelength is used to illuminate the sample, resulting in the emission of photons of a characteristic wavelength that can be detected by the sensor and further analyzed. In order to obtain optimal signals of the phenomena studied, a proper selection of excitation and emission filters as well as detectors with the spatial and temporal resolution needed must be chosen [63, 81].

Chapter 7 discusses the setup designed from a Langendorff perfusion system adapted to obtain optical mapping measurements of cardiac optogenetic experiments for this project and in Chapter 5 the technical details are also described. In optogenetics, the heart is stimulated by light. Therefore the setup is capable of stimulating the heart at certain wavelength and exciting the voltage-sensitive dye at different wavelength. Cardiac optogenetics will be described in detail in the following chapter.

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3.3. Arrhythmia treatment and the opportunity for optogenetics

Treatments will depend on the type of arrhythmia the patient is presenting, but there are generally three types of treatments: electric therapy, antiarrhythmic drugs and ablation [31, 37]. Antiarrhythmic drugs are mainly used in the cases of recurrent symptomatic arrhythmias.

Examples are beta blockers and ion channel blockers. However, in some cases antiarrhythmic drugs can worsen arrhythmias and by having proarrhythmic effects or result toxic for the patient [37, 16]. Electrical therapy, in the case of an implantable cardioverter-defibrillator, aids in the prevention of sudden cardiac death and can decrease the mortality of high-risk patients. Nonetheless, there is a risk of an inappropriate shocks being delivered [8]. And shocks (appropriate or inappropriate) are not free of risks. They can cause unbearable pain, damage to the myocardial tissue, psychological discomfort and increase the mortality of the patient [7, 52, 47, 37].

Optogenetics is a biological technique that enables light-induced depolarization of cells, and therefore has a great potential in the treatment of arrhythmias. Even though its use in the clinics still has different hurdles to face [69], the technique can be used as a tool to investigate the initiation and termination of arrhythmias at a spatiotemporal resolution uncomparable to the resolution offered by drugs or electrical therapy and with the promise of non-harming control of cardiac tissue. The next chapter is focused on describing optogenetics in the cardiac field. From the first findings in single cells and cell cultures to optogenetic cardioversion and its mechanisms.

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4. Cardiac Optogenetics

4.1. Introduction

The word “optogenentic” was introduced by Deisseroth in 2006 as a combination of optics, genetics and bioengineering in order to control and study neural circuits [22]. Nowa- days, optogenetics is used to control varied biological mechanisms through the expression of light-sensitive proteins.

In optogenetics, light sensitivity can be inscribed in cells and tissues, originally in the form of ion channels and pumps. However, the optogenetics toolbox now includes diverse proteins that with a shine of light of a specific wavelength can be used to control different cellular and sub-cellular functions [90, 49, 76]. Potential applications of optogenetics lie in neuroscience, oncotherapy, cardiovascular diseases, diabetes therapy, gene editing and other medical fields [95].

There are four main steps in order to apply optogenetics as a method to study living organisms [64]:

1. Selection or creation of the light activated proteins. The first channels to be tested were directly taken from algae. However, the advances in bioengineering have led to creation and modification of different channels and other proteins. Regarding ion channels, the range of applications has been amplified thanks to the manipulation of their properties.

Some of these are wavelength sensitivity, peak and steady current, delay from stimulation to activation, recovery time after activation and the sensitivity of photocurrent to changes in light intensity among others [55]. A clear example is ChR2-H134R -a mu- tant of ChR2 with greater steady current- which is most commonly used in the cardiac optogenetics field [58].

2. Gene delivery. There are mainly three methods to photo-sensitize cells; transfection, viral transduction or creating transgenic animals. A promoter can be used when applying viral therapy, giving optogenetics yet another possibility to make it cell or tissue specific. However, potential translation into the clinic represents a challenge and the most promising alternative at the moment appears to be viral gene delivery, which is still questionable for different safety reasons.

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4. Cardiac Optogenetics

3. Illumination. One of the key advantages of optogenetics compared to other methods of stimulation such as electrical or drugs is the spatio-temporal precision offered by light.

Illumination technologies can be at the same time the limiting factor and the facilitator.

The illumination source should depend on the type of control needed to be achieved.

From optical fibers [96], lasers, LEDs ([67]), micro-mirror devices ([19, 77, 50]) to microscopes [12], and micro-LEDs embedded in a 3D multifunctional integumentary membrane [94] each one will vary on spatio-temporal resolution, light-crafting capabilities and amount of energy delivered. Optical control of living systems such as the heart or cardiomyocytes offers a new level of interrogation capabilities that were impossible to achieve using electrical or drug stimulation.

Figure 4.1.:Optogenetic tools controlling membrane potential. The first applications of optogenetics were focused on changing the membrane potential of cells. (A)Ion channels such as Channelrhodopsin-2 (ChR2) and Channelrhodopsin-1 (VChR1) have been mainly used to depolarize the cells by allowing the entrance of cations, while ion pumps such as archaerhodopsin-3 (Arch) and NpHR hyperpolarize the membrane by either pumping cations to the exterior or anions to the interior of the cell. Moreover, these proteins are sensitive to(B)different wavelengths, therefore depolarizing and hyperpolarizing proteins could be combined to achieve a finer control of the membrane potential. Image obtained and modified with permission from [68].

4. Reading and interpreting the outcome. Some often used examples in the case of cardiac optogenetics can be the action potential during a patch clamp experiment, electrical signal such as the ECG using electrodes, or topological information of a monolayer or an isolated heart using optical mapping. The analysis of one or several outcomes will help better understand the electrophysiological behaviour of the heart.

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4.1. Introduction Since this doctoral thesis is focused on the understanding of optogenetic arrhythmia termination via global illumination, we will mainly focus on steps 3) and 4). For the first step it is of importance to understand the kinetics of the ion channel used in our model, Channelrhodopsin-2 (ChR2), and for the second step that we are using aα-MHC-ChR2 transgenic mouse model, which expresses ChR2 in>90 % of its cardiomyocytes.

4.1.1. Channelrhodopsin-2 (ChR2)

The identification of Channelrhodopsin-2 in the green algae Chlamydomonas rein- hardtii and its further cloning and characterization triggered the field of optogenetics as a technique to control excitable cells via light sensitive ion channels [57]. So far it has been the most important tool to study both neural and cardiac activity without the need of electrical or drug stimulation and it is also the optogenetic tool used in this doctoral work.

ChR2 is a non-selective cation channel with its highest affinity to H⁺, Na⁺, K⁺ and Ca₂⁺. Once it is illuminated, the opening of the channel will lead to a fast rise of its current (in approximately 200 µs). ChR2 uses retinal as its photosensing element. Since it is a passive membrane transport protein dependent on the electrochemical gradients, the current elicited with its opening will depend on the membrane potential.

Two different characteristic currents can be perceived; the peak current, reached on the first micro to milliseconds from the moment of illumination, and a steady-current which will have a lower amplitude and can be sustained for longer. The peak current of channel will also have a recovery time, therefore if a second light pulse is used to stimulate it, the current will depend on the time elapsed, and a full recovery can take up to several seconds something that will not affect the steady current coming afterwards. Closing of the channel will take approximately 10 ms to 20 ms. [57, 11, 26, 78].

4.1.2. Why optogenetic control?

Genetic targeting presents the possibility of discriminating the rest of the different cells and tissue that is not subject of optogenetic control or study. Light stimulation enhances even further the capabilities of spatial and temporal resolution and precision. With these new tools, a variety of scales can be chosen to control and study.

Moreover, electrical stimulation imposes an injection of current that affects the cell or tissue without considering the current electrophyiological condition of the target. An effect provided by an optogenetic stimulus will always be modulated by the ionic balances in and outside the cell, as well as by the other cells surrounding it, providing a sort-of feedback response that will manipulate the amplitude of the photo-current injected. Optogenetic control

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Figure 4.2.:Properties of Channelrhodopsin-2. (A)Channelrhodopsin-2 allows cations to flow with different affinity. The photo-current elicited is both (B) voltage- dependent and(C)light-dependent. When the membrane potential rests at greater negative value the current will be larger. Image obtained and modified with permission from [26]

.

generates bio-current, compared to super imposed electrical current that could lead to damage of the cells.

4.2. Optogenetic control of cardiac electrophysiology

The spatio-temporal control of the electrophysiology of the heart using optogenetics has been demonstrated with high precision at different scales. From milliseconds to seconds, cell level to organ level as well as different light intensities and illumination patterns, having an overview of all the capabilities in optogenetic control is of great importance to understand the outcome of such stimuli and to find an optimal solution for the control and termination of arrhythmia in the light-sensitive heart. It is well known that optogenetics offers high spatio- temporal precision but it is also important to consider that light intensity provides yet another dimension since different amounts of light will lead to different responses.

In one of the first computational models of cardiac optogenetics Abilez and colleagues [1] validated with experiments the ability to pace cardiomyocytes via optical stimulation at different frequencies and the effect of the light intensity on the photocurrent injected into the

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4.2. Optogenetic control of cardiac electrophysiology cells. When investigated using patch clamp, Channelrhodopsin-2 (ChR2) current reaches a peak during the first 20 ms of illumination and later decays to a steady-state current, both dependent on the intensity of the pulse applied. In a similar manner, lower intensities will result in longer delays of the action potential generation [11, 1]. Stimulating in a precise time- controlled fashion, optogenetic stimulation during the generation of an action potential can shape its morphology depending on the delay from the beginning of the action potential [92].

Optogenetic pacing of ChR2-expressing hearts will also lead to a 1:1 capture of the cardiac tissue, and for shorter pulses a higher amount of light will be required [11]. Moreover, the amount of energy needed to pace on the ventricles will be lower than in the atria [11], with the right ventricle being sensitive to the least amount of light [96].In vivospatial precision was corroborated using optical fibers to stimulate specific regions of the heart that were followed by varied responses depending on the stimulation place of ChR2 mice hearts [96, 17] (Fig.

4.6 E). In the same study, one more of the advantages of optogenetics was employed, as specific transgenic expression of ChR2 in either cardiomyocytes or the purkinje fibers was used to investigate the differences and susceptibilities of arrhythmia triggers between the two cell types and specific regions of the heart, proving that extrasystoles and ectopies can be generated by stimulating approximately ten fold less purkinje fibers than cardiomyocytes.

High spatio-temporal control in cardiac monolayers has seen further enhancement. Aided by technological advances, in this case a digital-micromirror-device (DMD), light was crafted in order to achieve precise control of excitation waves [15]. Direction of the waves was controlled by a synchronized series of light stimuli, first creating a one-sided conduction block with a long, high intensity pulse and during the period of refractoriness applying a next short low intensity pulse to generate a wave that would only travel the opposite direction of the block. Control of conduction velocity of the traveling waves was acquired by benefiting from the dimension offered by the manipulation of the light intensity. By applying a sub-threshold pulse (light at intensities that will not lead to an activation wave), cells’ membranes were brought close to the excitation point, therefore increasing the conduction velocity once a second light pulse triggering propagation was applied. Lastly, by imposing a spiral shaped light stimulus with opposite chirality, spiral wave chirality manipulation was achieved for the first time in excitable media.

At the moment of planning and designing optogenetic control, it is also of importance to consider the delivery method and how each one of these can lead to different spatial distri- butions of the light-sensitive cells, and at the same time, which kind of illumination is better suited to the distribution obtained [9].

The knowledge of all these features in optogenetic control of cardiac electrophysiology paved the way first to understand the mechanisms that can be used to terminate arrhythmias, and then to optimize arrhythmia control. The different methods and bases of optogenetic arrhythmia termination are described in the next section.

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Figure 4.3.:Optogenetic control of cardiac electrophysiology.Caption on next page.

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4.3. Optogenetic arrhythmia termination

Figure 4.3.:Optogenetic control of cardiac electrophysiology. Experimental and computational work has shown that the electrophysiological behaviour induced by ChR2 will depend on the intensity of the stimulus. (A)Photocurrents will be larger for higher intensities, both peak and steady-state currents(B). Additionally,(C)light stimulation of individual cells, cardiac monolayers or beating hearts will derive on temporal response and activation of the target, and on single cells(D)higher intensities will translate into faster triggering of action potentials. (E) Regional control was also proved using optical fibers to stimulate specific areas of the heart and confirmed on the ECG recordings. (F)Direction, speed and spiral chirality of excitation waves on cardiac monolayers was achieved using a micro-mirror device. Images obtained and modified with permission from [1] for A and C, [11]

for B and D, [96] for E, [15] for F.

4.3. Optogenetic arrhythmia termination

One of the most ambitious and desired goals in the field of cardiac optogenetics is the possibility of pain-free and therefore harmless arrhythmia termination. A potential application could be seen in an optogenetic implantable cardioverter-defibrillator (ICD). Patients implanted with ICD not only suffer from pain at the moment of the shocks, but also from depression and anxiety showing a reduction in their self-perceived quality of life, mainly due to the application of irregular shocks and the development of other symptoms that affect the patients mentally and physically [79, 89].

The first proof of optogenetics-based arrhythmia termination was shown on monolayers of neonatal rat atrial cardiomyocytes [6]. Spiral waves were electrically induced and then terminated by photo-stimulation of the Ca²⁺-permeable channelrhodopsin CatCh [45], which is also sensitive to blue light. Light pulses of 500 ms at 0.038 mW/mm²led to arrhythmia termination in all the cell cultures where CatCh was expressed. Spiral termination was attributed to the decrease in excitability by the light-induced depolarization that lead to meandering and collision of phase singularities with one another or with the boundaries (Fig 4.4).

This study demonstrated the potential of light induced arrhythmia control. However, a translation to clinical applications is still many challenges away. Two of the most immediate ones are related to the translation from a 2D culture model to a whole-heart model. First, arrhythmia behaviour is more complex in a heart and the physical boundaries such as the ones found in a 2D model are not there. And second, light-penetration can become an issue.

Bingen and colleagues relied on homogeneous illumination of the cardiomyocyte monolayers [6], therefore one can expect that almost all the cells received the same intensity. Achieving a stimulus as precise and as global as the one achieved by them is a practically impossible challenge, even without mentioning that simply achieving stimulation of all the cells in the heart could already be a challenge.

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Figure 4.4.:Effective light-induced spiral wave termination by CatCh expression. (A) Activation map of CatCh-transduced rat atrial cultures showing spiral arrhythmic activity before being optogenetically stimulated (left) and after 500 ms of homogeneous illumination, showing planar wave eletrcial propagation. (B)Rate of successful reentry termination for CatCh-transduced cultures (n=31) and cultures transduced only with eYFP (n=11, control). Image obtained and modified with permission from [6].

4.3.1. First “optical shocks” in small animal models

Nonetheless, in 2016 three different groups were able to revert arrhythmic heartsex-vivo into their normal rhythm via optogenetical control of their electrophysiology. A combination of two different animal models, using two versions of channelrhodopsins and also diverse illumination strategies opened the road into a new dimension of cardiac arrhythmia research.

While the works by Bruegmann and Crocini were done on ChR2-transgenic mice hearts [12, 19], Nyns and colleagues stimulated red-activatable channelrhodopsins (ReaChR) in rat hearts treated with the injection of adeno-associated virus [61].

Since the work of this thesis was performed using ChR2-transgenic mice hearts, the results obtained in the same model were actually used to set the foundations of the research performed and will be more broadly discussed. Using the K_{AT P} channel opener pinacidil, Bruegmann et al. were able to induce arrhythmia in the Langendorff-perfused mouse heart, and by illumination of the anteroseptal epicardium able to perform optogenetic cardioversion on the beating heart. K_{AT P} are potassium channels sensitive to adenosine triphophate (ATP) and have a protective role during ischaemia [28]. Their opening leads to the shortening of the action potential duration, facilitating the initiation of arrhythmias.

Compared to optogenetic pacing, cardioversion demands increased amounts of energy, which in optogenetics can be translated into into higher intensities, longer pulses and larger areas to illuminate. In this experiments it was also demonstrated that for the same reason length, intensity and area have an influence in the efficiency of the termination attempt (Fig

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4.3. Optogenetic arrhythmia termination

Figure 4.5.:Optogenetic defibrillation of the mouse heart by illumination of the epicardial surface. Stimulation of Langendorff-perfused hearts in arrhythmic state using a 4-pulse protocol shows that efficiency depends on(A)pulse length (n=7), (B) area illuminated (n=9) and (C) the intensity delivered to the cardiac tissue (n= 5). For the parameters tested the pulse length maximum efficiency appears to be reached between 300 and 1000 ms, while the intensity saturates at 1.0 mW/mm². (D) Arrhythmia was induced benefiting from the effects of pinacidil via electrical burst pacing and the stimulated protocol consisted of 4 light pulses of identical characteristics. Data presented as mean ±S.E.M. Each data point represents the termination rate in one heart. Image obtained and modified with permission from [12]

.

4.5). In order to obtain a higher efficiency, the number of pulses was increased to 4, which allowed them to terminate at a rate of 97 % with stimuli of 0.4 mW/mm² lasting 1 second on an area of 143 mm². Additional experiments of clinical relevance performed by Bruegmann and colleagues in the same work demonstrated the feasibility of optogenetic cardioversion in hearts with induced acute myocardial infarction and in a different scenario the termination of arrhythmia wild type mice heart one year after gene transfer via AAV injection.

The approach of Crocini et al. was different. Even though they also looked to optogenetically terminate arrhythmia they aimed to take full advantage of the spatio-temporal benefits of

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using optogenetics. In order to do this they designed an imaging and stimulation setup using a macroscope and a laser scanning system based on acousto-optics deflectors. This allowed them to “draw” different illumination patterns on the surface of the heart (Fig 4.6).

Figure 4.6.:Shaped illumination in optogenetic defibrillation. (A)Success rates for different shapes tested. The intensity of the triple barrier is 10 mW/mm² and whole LV was illuminated with an intensity of 0.5 mW/mm². Data presented as mean± S.E.M. (B)The triple barrier was tested using different intensities as well as two pulse durations. The highest success rate obtained using 40 mW/mm²and 10 ms was also compared to three barriers placed in a different position terminating only 36% of the attempts. Image obtained and modified with permission from [19].

Since the arrhythmias generated in their ChR2-mice hearts consisted of a re-entrant spiral covering the left ventricle they opted to use a triple barrier stimulation shape (Fig 4.6A) in order to depolarize the ChRh2-cardiomyocytes on this specific areas and generate conduction blocks along the path of the tachycardia.

With this concept, thetriple barrier terminated arrhythmia at a rate of 98% covering a total area of 0.45 mm² compared to Bruegmann’s 143 mm² using a series of 10 pulses of 10 ms at an intensity of 40 mW/mm², which is 100-fold greater than the intensity used in the first approach.

As mentioned before, both groups used different manners in order to end arrhythmic behavior using optogenetics for the first time in the beating heart [20]. An advantage from the first group was the ability to terminate non-specific arrhythmia and do it at evidently lower intensities. On the other side it required 4 pulses of 1000 ms with “off” breaks of 1-5 seconds in between. Altogether this can be a great amount of time considering the physiology and heart rate of the mouse heart.

The second group, which needed only a total of 100 ms of stimulation and 1000 ms

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4.3. Optogenetic arrhythmia termination to complete the termination protocol suffered a compensation in energy by increasing the intensity up to 40 mW/mm², which would need validation of no harm induced on the cardiac tissue since phototoxicity is one of different challenges to be faced by cardiac optogenetics [69].

Figure 4.7.:Global illumination of the epicardial surface.A setup composed of three LEDs surrounding the heart enables simultanous illumination of the whole heart which could resemble electrical defibrillation. Image obtained and modified with permission from [67].

The most important part of this doctorate work was the aim to use yet a different approach that would tackle the disadvantages of the two methods previously described, which are the need of multiple and long stimulation pulses or the need of very high intensities. There- fore a stimulation setup illuminating the complete surface area of the heart was designed (Fig 4.7). Apart from allowing us to reduce the intensity needed and the length of the pulses, it would also help us understand how arrhythmia behave in a scenario similar to the application of electrical shocks, where the complete heart is stimulated. Chapter 7 is dedicated to the results and findings obtained with this aim.

4.3.2. Mechanisms of optogenetic cardioversion

Sasse and colleagues described optogenetic cardioversion via depolarization using ion channels as an outcome from two possible mechanisms; conduction block or filling of the excitable gaps [76]. An excitable gap is the area of the heart or tissue that has been depolarized and has had enough time to recover from the refractory period, and therefore is again excitable (Fig 4.8 A).

During conduction block certain volume, thickness or area of the cardiac tissue is il-

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Figure 4.8.:Mechanisms of cardioversion by optogenetic depolarization. (A) After depolarization of the cardiac tissue occurs (red), it cannot be excited again for a period of time while it recovers (yellow), and lastly once it is recovered it is considered an excitable gap again (green). (B)Arrhythmic spiral wave at different time points, where the wave front travels and behind it the cardiac media shows refrac- torines for a period of time before becoming excitable again. (C)A conduction block consists of continuously illuminating an area of the tissue in order to keep it in a refractory period until the next arrhythmic wave front rout is blocked by the illuminated area. (D)Filling the excitable gap consists in illuminating part of the tissue that can be excitable in order to generate a new wave that would clash with the arrhythmic wave leading to its annihilation. Image obtained with permission from [76].

luminated and brought to an unexcitable state long enough to block or disrupt the activation path of the arrhythmia, therefore halting its activity (Fig 4.8 C). In this case, the amount of volume stimulated should be enough to properly create the block since a partial block can still allow circulation of the arrhythmic wave through non-excited or deeper areas of the ventricles [88, 41]. Filling of the excitable gap consists of stimulating an available region of the tissue in order to generate a depolarization wave that can collide with the arrhythmia and lead to the termination of both (Fig 4.8 D).

Creating a conduction block requires continuous stimulation of the arrhythmic wave path, hence illumination of the deeper layers of the heart will be of importance and can be a limiting factor. Differently, filling the excitable gap can be accomplished with a lower amount of light but it requires timing and positioning of the gap.

4.3.3. Determinants in optogenetic cardioversion

As previously mentioned, Nyns and colleagues accomplished optogenetic cardioversion on rat hearts using the red-activatable channelrhodopsins (ReaChR). More interestingly, they

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Arrhythmia termination using Global Optogenetic Stimulation in ChR2 mice hearts